Modified bacterial cellulose

ABSTRACT

This invention provides a bacterial cellulose comprising ribbon-shaped microfibrils having a thickness of 10 to 100 nm and a width of 160 to 1000 nm or a bacterial cellulose comprising ribbon-shaped microfibrils having a thickness of 10 to 100 nm and a width of 50 to 70 nm. The former bacterial cellulose can be produced by culturing cellulose-producing bacteria in a culture medium containig a cell division inhibitor, and the latter can be produced by culturing the bacterium in a culture medium containing an organic reducing agent. The bacterial cellulose is modified from conventional bacterial cellulose in the major axis, and is improved in Young&#39;s modulus, etc.

BACKGROUND OF THE INVENTION

This invention relates to bacterial cellulose (BC) of whichribbon-shaped microfibrils are artificially modified to improve Young'smodulus and a method of producing the same.

The bacterial cellulose can be used as various industrial materials,clothing materials, materials for medical supplies, functionalmaterials, materials for foods and so on.

It is known that Acetobacter xylinum ATCC 23769 produces a mat-shapedcellulose which can be used for medical pads (Japanese Patent KOKAI59-120159). It is also known that Acetobacter aceti subsp. xylinum ATCC10821, etc. produce bacterial cellulose composed of ribbon-shapedmicrofibrils (U.S. Pat. No. 4,742,164). The size of the ribbon-shapedmicrofibril is said to be 20 to 50 nm (Ed. by Tokyo Techno. ForumSecretariat, "Jinrui to Bio (Humanity and Bio)", P329, 1993, YomiuriNippon Television (enter) which may be measured without discriminationof the major axis (width and the minor axis (thickness).

The bacterial cellulose is produced as floc or suspended matter in aform of sheet, dispersion, grain or the like by static culture oraeration agitation culture which effects entangling of fibers. However,although the above macroscopic variation occurs, ribbon-shapedmicrofibril and properties of the bacterial cellulose are substantiallynot varied.

Structure and properties of bacterial cellulose are slightly differentaccording to the type of bacterium. However it has not been reported toproduce modified bacterial cellulose by changing the form ofcellulose-producing bacteria artificially to vary ribbon-shapedmicrofibrils.

SUMMARY OF THE INVENTION

An object of the invention is to develop a bacterial cellulose, whereinthe major axis (width) of ribbon-shaped microfibril is varied, andvarious properties, especially Young's modulas are improved.

The inventors investigated in order to achieve the above object, andfound that a modified bacterial cellulose wherein ribbon-shapedmicrofibrils are varied can be obtained by adding a cell divisioninhibitor or an organic reducing agent to a culture medium which inducesvariation of the shape of cellulose - producing bacteria, and thatproperties, especially Young's modulus, are improved compared withconventional bacterial cellulose.

Thus, the present invention provides, bacterial cellulose comprisingribbon-shaped microfibrils having a thickness of 10 to 100 nm and awidth of 160 to 1000 nm, a method of producing bacterial cellulose whichcomprises culturing cellulose-producing bacteria which produce thebacterial cellulose extracellularly in a culture medium containing acell division inhibitor, and recovering the bacterial cellulose producedin the culture medium, and further the present invention providesbacterial cellulose comprising ribbon-shaped microfibrils having athickness of 10 to 100 nm and a width of 50 to 70 nm, and a method ofproducing bacterial cellulose which comprises culturingcellulose-producing bacteria which produce the bacterial celluloseextracellularly in a culture medium containing an organic reducingagent, and recovering the bacterial cellulose produced in the culturemedium.

In the invention, a section of a ribbon-shaped microfibril perpendicularto the growth direction (lengthwise direction) is assumed a rectangle,and one side is called the width or the major axis and the other side iscalled the thickness or the minor axis. In general, the length of themajor axis is longer than the minor axis.

The microfibril of bacterial cellulose of the invention can bediscriminated from conventional microfibrils by measuring the length ofeach major axis and minor axis using an electron microscope of atomicforce microscope.

It is seemed that the shape or the number of cellulose secretion portvaries due to the variation of the shape of the bacterium, and thereby,the shape of microfibril is varied. From experimental results, bacterialcellulose produced by long cell bacteria has a higher clarity than shortcell bacteria, and the results suggest that the cellulose produced bylong cell bacteria is in a more dense state. This is also supported bythe observation of bacterial cellulose using a scanning electronmicroscope (SEM) and an atomic force microscope, and therefore, thecellulose produced by long cell bacteria has a more dense layerstructure. In the conventional cellulose produced by normal bacteria,portions where cellulose is deposited in a helicoidal (cholesteric) formare observed, but the portions are not present in the cellulose producedby long cell bacteria. As to crystal width, it is considered that thecellulose produced by long cell bacteria is, although slightly, greaterthan the cellulose produced by normal bacteria in all lattice planes. Inall bacterial cellulose, 0.6 nm lattice planes are oriented against filmface, the cells are greater, the orientation degree is higher. In theobservation of bacterial celluloses using a transmission electronmicroscope (TEM), the width of ribbon-shaped microfibril produced bylong cell bacteria is greater than that produced by normal bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS.

FIG. 1 is a photograph of an atomic force microscope showing a shaped ofcellulose fiber and a cellulose-producing bacterium which was culturedwithout cell division inhibitor and organic reducing agent.

FIG. 2 is a section taken on line A-B of FIG. 1 which was judged to be aminor axis portion.

FIG. 3 is a section taken on line C-D of FIG. 1 which was judged to be amajor axis.

FIG. 4 is a photograph of an optical microscope (×1000) showing a shapedof a cellulose-producing bacterium which was cultured in a 0.3 mMchloramphenicol-added culture medium.

FIG. 5 is a photograph of an optical microscope (×1000) showing a shapedof a cellulose-producing bacterium which was cultured in a culturemedium to which chloramphenicol was not added.

FIG. 6 is a photograph of an atomic force microscope showing a shaped ofcellulose fiber and a cellulose-producing bacterium which was culturedin a 0.3 mM chloramphenicol-added culture medium.

FIG. 7 is a photograph of an atomic force microscope showing a shaped ofcellulose fiber and a cellulose-producing bacterium which was culturedin a 1.0 mM dithiothreitol-added culture medium.

DETAILED DESCRIPTION OF THE INVENTION

The bacterial cellulose of the invention comprises ribbon-shapedmicrofibrils having a minor axis of 10 to 100 nm and a major axis of 160to 1000 nm or 50 to 70 nm. The inventors cultured cellulose-producingbacteria (Acetobacter pasteurianus FERM BP-4176) in a culture mediumwithout containing cell division inhibitor and organic reducing agent,and the size of the microfibrils of the bacterial cellulose wasmeasured. As a result, the microfibril had a minor axis of 10 to 100 nmand a major axis of 80 to 150 nm. Accordingly, the bacterial celluloseof the invention is clearly different from conventional bacterialcellulose.

The minor axis of microfibrils is, in general, 55 to 95 nm, occasionallysmaller size, e.g. 25 nm, irrespective of the bacterial cellulose of theinvention obtained by culturing in a culture medium containing a celldivision inhibitor or an organic reducing agent or conventionalbacterial cellulose obtained by culturing in a culture medium notcontaining cell division inhibitor and organic reducing agent.

On the other hand, the major axis of the microfibrils of the bacterialcellulose obtained by culturing in a culture medium containing a celldivision inhibitor is, in general, 160 to 700 nm, particulary 170 to 600nm, occasionally longer size, e.g.1000 nm. That is, the major axis isconsiderably greater compared with conventional major axis of 80 to 150nm. When a culture medium contains a cell division inhibitor,cellulose-producing bacteria are lengthened, and it is observed that aplurality of single chains are adhered to each other to form a bundle.The bundle can be deemed single chain, and accordingly, the major axisbecomes considerably longer than conventional one. The ratio of majoraxis:minor axis is about 2.8:1.0 to 8.1:1.0, particularly, 3.0:1.0 to6.0:1.0. In the case of conventional microfibrils, the ratio of majoraxis/minor axis is 1.6:1.0 to 2.7:1.0.

In the case of the bacterial cellulose obtained by culturing in aculture medium containing an organic reducing agent, the major axis ofthe microfibrils is, in general, 50 to 70 nm, and it is difficult todiscriminate the major axis and the minor axis. It is considered to becaused by shortening of bacterial cell. The ratio of major axis: minoraxis is about 0.9:1.0 to 1.5:1.0, particularly, 1.2:1.0 to 1.5:1.0.

The bacterial cellulose is characterized by the improvement in Young'smodulus which is increased by 30% or more compared with conventionalbacterial cellulose obtained in a culture medium not containing celldivision inhibitor and organic reducing agent. The Young's modulus ofthe bacterial cellulose having a major axis of microfibril of 160 to1000 nm is about 13 to 20 GPa, particularly about 16 to 20 GPa, and theYoung's modulus of the bacterial cellulose having a major axis ofmicrofibril of 50 to 70 nm is about 14 to 19 GPa, particularly about 15to 18.5 GPa. The effect of the improvement in Young's modulus isremarkable in the case of the cellulose obtained by culturing in aculture medium containing a cell deivision inhibitor, particularly,pyridone carboxylic acid based agents. Because major axis of themicrofibrils of the bacterial cellulose is considerably lengthened inorder to lengthen bacterial cell remarkably. The elongation at ruptureof the bacterial cellulose having a major axis of microfibril of 160 to1000 nm is about 0.9 to 2.1%, particularly about 1.4 to 1.8%, and theelongation at rupture of the bacterial cellulose having a major axis ofmicrofibril of 50 to 70 nm is about 0.9 to 2.0%, particularly 0.9 to1.5%.

As the chemical components of the bacterial cellulose, there arecellulose, cellulose as a main chain and containingheteropolysaccharides or α-, β- , etc., glucans. In the case ofheteropolysaccharides, the constituent components, other than cellulose,are hexose, pentose and organic acids, etc., such as mannose, fructose,galactose, xylose, arabinose, ramnose, uronic acid, etc. Thesepolysaccharides may be single substances; alternatively, two or morepolysaccharides may coexist.

Microorganisms that produce such bacterial cellulose are notparticularly limited, and include, Acetobacter pasteurianus ATCC 23769,FERM BP-4176, Acetobacter aceti, Acetobacter xylinum, Acetobacterrancens, Sarcina ventriculi, Bacterium xyloides and bacteria belongingto the genus Pseudomonas, the genus Agrobacterium, the genus Rhizobium,etc.

It is important that the culture medium in which cellulose-producingbacterium is cultured contains a cell division inhibitor or an organicreducing agent.

The cell division inhibitor includes chloramphenicol based antibiotics,such as chloramphenicol, protein synthesis inhibitors, such astetracycline, puromycin and erythromycin, organic compounds havingβ-lactamase inhibiting ability, such as thienamycin, pyridone carboxylicacid based agents, such as nalidixic acid, promidic acid, pipemidicacid, oxolinaic acid, ofloxacin, enoxacin, and so on. A suitableconcentration of the cell division inhibitor is, in the case ofchloramphenicol, 0.01 to 5.0 mM, preferably 0.05 to 1.0 mM, morepreferably 0.1 to 0.5 mM, and in the case of nalidixic acid, 0.01 to 1.0mM, preferably 0.05 to 0.3 mM, more preferably 0.1 to 0.2 mM. In aconcentration less than the lower end, i.e. 0.01 mM, modification ofbacterial cellulose is insufficient, and in a concentration exceedingthe upper end, i.e. 5.0 mM or 1.0 mM, growth of bacteria is greatlyinhibited.

The organic reducing agent includes dithiothreitol, 2-mercaptoethanoland so on. A suitable concentration of the organic reducing agent is, inthe case of dithiothreitol, 0.01 to 5.0 mM, preferably 0.2 to 3.0 mM,more preferably 0.5 to 2.0 mM. In a concentration less than the lowerend, modification of bacterial cellulose is insufficient, and in aconcentration exceeding the upper end, growth of bacterium is greatlyinhibited.

The other components of the culure medium may be similar to a knownmedium used for culturing the aforementioned bacteria. That is, theculture medium contains a carbon source, a nitrogen source, inorganicsalts and, if necessary, organic minor nutrients such as amino acids,vitamins, etc. As the carbon source, glucose, sucrose, maltose, starchhydrolysate, molasses, etc., can be used, but ethanol, acetic acid,citric acid, etc., may also be used singly or in combination with theabove-desribed sugars. As the nitrogen source, organic or inorganicnitrogen sources such as ammonium salts, e.g. ammonium sulfate, ammoniumchloride, ammonium phosphate, etc., nitrates, urea, peptone or the likecan be used. Inorganic salts are minor phophates, magnesium salts,calcium salts, iron salts, manganese salts, etc. As organic nutrientsamino acids, vitamins, fatty acids, nucleic acids, etc. are used.Furthermore, peptone, casamino acid, yeast extracts, soybean proteinhydrolysates, etc., containig these nutrients may be used. When usingauxotrophs requiring amino acids, etc., for growth, it is necessary toadd required nutrients.

Cultivation method is also not limited, and may be static culture,agitation culture (aeration agitation culture, shaking culture,oscillation culture, air lift type culture) or the like.

The culture conditions may be conventional: for example, at a pH of 3 to9, preferably 3 to 7, and at a temperature of 1 to 40° C., preferably 25to 30° C., culture is performed for 1 to 100 days. In the case of staticculture, bacterial cellulose is dispersed in the culture solution in theinitial stage, and accumulated as a surface layer in a gel form in thelater stage.

The gel is withdrawn and washed with water, if necessary. Depending uponthe intended use of the gel, the washing water may contain chemicalssuch as sterilizers, pre-treating agents, etc.

After washing with water, the gel is dried or kneaded with othermaterials follwed by drying. The drying may be carried out by any mannerbut within the temperature range wherein bacterial cellulose is notdecomposed. Since the bacterial cellulose is composed of fine fibershaving many hydroxyl groups on their surfaces, it is possible to losefiber form due to coadhesion of fibers during drying, Accordingly, whenbacterial cellulose is used with utilizing fine fiber shape, freezedrying and critical point drying are preferable in order to avoid thecoadhesion of fine fibers.

It is preferred that the bacterial cellulose is of structure in whichthe microfibrils are intertwined, in order to enhance the dynamicstrength such as Young's modulus, etc. For this reason, an effectivemethod comprises pressing the gel, harvested from the culture, from theorthogonal direction, squeezing most of the free water off and thendrying it. It is appropriate that the squeezing pressure beapproximately 1 to 10 kg/cm². By this press squeezing, the celluloseafter drying is orientated along the press squeezing direction.Furthermore, by stretching in one direction while applying pressure,e.g. by performing a rolling operation, the cellulose after drying isorientated also in the rolling direction, in addition to the presssqueezing direction. Pressing apparatuses can be appropriately chosenfrom commercially available machines,

On the other hand, it is also effective to macerate the bacterialcellulose, in order to increase the dynamic strength. Maceration may becarried out by using a mechanical shearing force. The bacterialcellulose can easily be macerated with, for example, a rotary macerator,a mixer, etc. It is also effective to conduct the aforesaid presssqueezing after maceration.

The bacterial cellulose can be formed into various shapes such assheet-liked shapes, yarn-like shapes, cloth-like shapes, solid-likeshapes, etc.

In the case of molding into a sheet-like form, the bacterial celluloseis, if desired, macerated and then formed into a layer, which issqueezed under pressure, if desired, and then dried. By press squeezing,a planar-orientated sheet is obtained; by further rolling, a sheet notonly planar-orientated but also uniaxially orientated can be obtained.

It is desired that the drying of the sheet, macerated and/or presssqueezed, are carried out after fixing it to a suitable support. Byfixing it on a support, the degree of planar-orientation is furtherenhanced and a sheet having a large dynamic strength can be obtained. Assupports, plates, e.g. glass plates, metal plates, etc., having, forexample, a net structure, can be used. Any drying temperature can beused as long as the temperature is within a range where the cellulose isnot decomposed. In addition to heat drying, freeze drying can also beused.

The thickness of the sheet depends upon its intended use, but isgenerally about 1 to 500 microns.

The sheet may contain various additives. For example, by incorporatingsolutions (aqueous or nonaqueous), emulsion, dispersions, powders,melts, etc. of various polymer materials, one or more of strength,weatherproofness, chemical resistance, water resistance, waterrepellency, antistatic properties, etc., can be imparted to the sheet,depending upon the properties of the additives. By incorporating metalssuch as aluminium, copper, iron, zinc, etc., or carbon in a powdery formor fibre form, electroconductivity and thermal conductivity can beincreased. Further, by incorporating inorganic materials such astitanium oxide, iron oxides, calcium carbonate, kaolin, bentonite,zeolite, mica, alumina, etc., the heat resistance, insulatingproperties, etc., can be improved or smoothness can be imparted to thesurface, depending upon kind thereof. By incorporating low molecularweight organic materials or adhesives, the strength can be furtherincreased. The sheet may be coloured with colouring agents such asphthalocyanine, azo compounds, indigos, safflowers, etc. For coloration,various paints, dyes and pigments can be used in addition thereto. Byincorporating medicines or sterilizers, the sheet can also be utilizedas a medical sheet.

These kneadings and additives are incorporated in an appropriate amountnot exceeding 97% capable of imparting the desired physical properties.The time of the incorporation is not limited, and they may beincorporated in the bacterial cellulose gel or a macerated productthereof; alternatively, they may be incorporated after press squeezingor after drying. Furthermore, they may be incorporated in the culturemedium or culture on some occasions. The method of incorporation may beby impregnation, as well as by mixing.

On such a sheet can also be laminated a layer of other material. Thelaminate can be appropriately chosen depending upon the intended purposeof the sheet. The laminate can also be chosen from the aforesaidkneadings or additives; for example, various polymer materials can becoated onto the sheet to impart waterproofness to the sheet.

In the case of paper, the bacterial cellulose gel is macerated, thensubjected to paper making and then drying, whereby paper obtained has anexcellent tensile strength, resistance to expansion, etc as well ashaving a high elasticity and a high strength. The paper is chemicallystable and excellent in water absorbance and aid permeability. In thiscase, ordinary additives, treating agents, etc., used for paper makingcan be utilized and kneadings and additives can also be appropriatelychosen from the aforesaid substances and incorporated into the paper.

The sheet formed of the bacterial cellulose is usable as an acousticdiaphragm having excellent properties.

Other uses are disclosed in U.S. Pat. No. 4,742,164, etc.

EXAMPLES Example 1

The culture medium used was composed of 50.0 g/l sucrose, 5.0 g/l "TotalAmino Acid" (Ajinomoto Co., Inc.), 0.2 g/l phytic acid, 2.4 g/lmagnesium sulfate and 1.0 g/l ammonium sulfate (pH 5.0).

Seed culture was carried out by placing 20 ml of the above culturemedium in a 100 ml flask with baffle, inoculating Acetobacterpasteurianus FERM BP-4176, and then culturing at 25° C. for 3 days withstirring at 200 rpm. The culture medium was crushed by a blender, andadded to a main culture medium having the above composition in aconcentration of 2% seed culture.

The main culture was carried out by static culture at 25° C. During theculture, culture solution and bacterial cellulose were withdrawn, andthe morphology of bacteria was observed by an optical microscope, anelectron microscope and an atomic force microscope.

Six main culture media were used, and nalidixic acid (NA) was addedthereto in a concentration of 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM or 1.0 mMexcept one medium to which NA was not added.

As a result, production of bacterial cellulose was inhibited withincreasing the NA concentration. For example, the shape of the bacteriumafter cultured in the medium containing 0.1 mM NA and that cultured inthe medium not containing NA for 2 days were compared by taking each anoptical microscope photograph (×1000). As a result, in the case of 0.1mM NA, the shape of bacterium was varied and lengthened 2 to 4 timescompared with no addition of NA.

The ribbon-shaped microfibrils produced in NA-added media were observedby the electron microscope and the atomic force microscope, and foundthat the major axes (width) was great, e.g. 170 nm, 340 nm, 430 nm, 590nm, etc., but the minor axes (thickness) were in the range of 10 to 100nm, e.g. 25 nm, 30 nm, 60 nm, 90 nm etc. On the other hand, theribbon-shaped microfibrils produced in no NA added medium had a majoraxis (width) of 82 nm, 107 nm, etc and a minor axis (thickness) in therange of 10 to 100 nm, and significant variation was not observedcompared with NA added medium concerning the minor axis.

A part of cellulose gel after culturing 2 days was harvested, and put ona cover glass. The cover glass was allowed to stand at room temperaturefor 10 to 20 minutes to dry the surface naturally. The cellulose gel wasobserved by an atomic force microscope ("SPM-9500", Shimazu Seisakusho),and an example is shown in FIGS. 1-3. FIG. 1 is an atomic forcemicroscope photograph of a cellulose-producing bacterium grown in aculture medium not containing cell division inhibitor and organicreducing agent which is secreting bacterial cellulose. An operationseeks the narrowest part and the widest part of a cellulose fiberproduced from the bacterium, and lines for image analysis are drawn atthose parts in the direction perpendicular to the fiber lengthwisedirection on the image displayed on a display of a computer connected tothe atomic force microscope, and the shape (section) in the directionperpendicular to the fiber lengthwise direction is displayed (FIGS.2,3). Then, the operator operates the computer to display each length.The minor axis indicated by A-B line was ca. 86 nm, and the major axisindicated by C-D line was ca. 123 nm.

After culturing for 40 days, the bacterial cellulose gel was taken out,and washed with running water, alkali, and then running water,succesively. The washed bacterial cellulose was pressed into sheet andproperties were measured as to 0.1 mM NA, 0.2 mM and no NA.

Each bacterial cellulose sheet was punched into dumbbell pieces of JISstandard No.3 having a width of 1.0 cm and a length of 2.0 cm, and usedas test pieces. After measuiring the thickness of each test pieces, andits strength was measured by a tensile tester "Tensilon RTM-600 Type"(Orintec Corp.) with drawing at a rate of 20 mm/min, the results areshown in Table 1.

                  TABLE 1                                                         ______________________________________                                                     Mean           Mean          Mean                                                                            Thick- Thick- Young's Elastic                                               Elongation Elongation                 NA ness ness Modulus Modulus at Rupture at Rupture                            (mM) (μm) (μm) (GPa) (GPa) (%) (%)                                    ______________________________________                                        0.10 33      32      19.4   19.4   1.51   1.79                                   35  19.7  1.90                                                                31  19.5  2.02                                                                29  19.2  1.72                                                               0.20 31 34 16.4 16.1 1.78 1.88                                                 35  18.2  2.12                                                                34  13.9  2.03                                                                35  15.8  1.58                                                               0   25 38 11.8 12.4 1.82 1.80                                                  44  11.3  2.22                                                                54  14.1  1.53                                                                32  12.3  1.62                                                             ______________________________________                                    

As shown in Table 1, the sheets obtained by culturing in 0.1 mM NAmedium and in 0.2 mM NA medium varied in their properties, and Young'smodulus was improved compared with the sheet obtained by culuring in noNA medium.

Example 2

Acetobacter pasteurianus FERM BP-4176 was cultured in static culture,and the culture solution and bacterial cellulose were withdrawn, and theshape of bacteria was observed by the optical microscope, the electronmicroscope and the atomic force microscope, similar to Example 1, exceptthat chloramphenicol was used instead of nalidixic acid.

That is, six main culture media having the aforementioned compositionwere used, and chloramphenicol (CP) was added thereto in a concentrationof 0.1 mM, 0.2 mM, 0.3 mM. 0.5 mM or 1.0 mM except one medium to whichCP was not added.

As a result, the length of the cellulose-producing bacterium increasedwith increasing the CP concentration up to 8 to 12 times as long as thebacteria cultured in no CP medium.

As an example, the shape of bacterium cultured in the 0.3 mM CP mediumfor 2 days taken by the optical microscope (×1000), and shown in FIG. 4,and that cultured in no CP medium for 2 days is shown in FIG. 5.

The CP ribbon-shaped microfibrils produced in NA-added media wereobserved by the electron microscope and the atomic force microscope, andfound that the major axes (width) was great, e.g. 160 nm, 330 nm, 450nm, 570 nm, 690 nm, etc., but the minor axes (thickness) were in therange of 10 to 100 nm. On the other hand, the ribbon-shaped microfibrilsproduced in no CF added medium had a major axis (width) of 82 nm, 107nm, etc and a minor axis (thickness) in the range of 10 to 100 nm, andsignificant variation was not observed compared with CP added mediumconcerning the minor axis.

After culturing 40 days, the bacterial cellulose produced was made intoa sheet, and properties of the sheets obtained from 0.2 mM CP, 0.3 mM CPor no CP were measured, similar to example 1. The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                                     Mean           Mean          Mean                                   Thick- Thick- Young's Elastic Elongation Elongation                          CP ness ness Modulus Modulus at Rupture at Rupture                            (mM) (μm) (μm) (GPa) (GPa) (%) (%)                                    ______________________________________                                        0.20 35      36      18.2   19.3   1.63   1.29                                   37  20.2  1.26                                                                35  19.4  1.03                                                                36  19.6  1.22                                                               0.30 34 35 13.4 16.5 1.93 1.40                                                 37  17.8  1.42                                                                35  14.5  1.28                                                                34  18.2  0.98                                                               0   25 38 11.8 12.4 1.82 1.80                                                  44  11.3  2.22                                                                51  14.1  1.53                                                                32  12.3  1.62                                                             ______________________________________                                    

As shown in Table 2, the sheet obtained by culturing in 0.2 mM 0.3 mM CPmedium varied in its properties, and Young 's modulus was improvedcompared with the sheet obtained by culuring in no CP medium.

Example 3

Acetobacter pasteurianus FERM BP-4176 was cultured in static culture,and the culture solution and bacterial cellulose were withdrawn, and theform of bacteria was observed by the optical microscope, the electronmicroscope and the atomic force microscope, similar to Example 1, exceptthat chloramphenicol was used instead of nalidixic acid.

That is, four main culture media having the aforementioned compositionwere used, and dithiothreitol (DTT) was added thereto in a concentrationof 0.5 mM, 1.0 mM or 2.0 mM except one medium to which DTT was notadded.

As a result, the length of the cellulose-producing bacterium decreasedwith increasing the DTT concentration.

As an example, the shape of bacterium cultured in the 1.0 mM DTT mediumfor 2 days taken by the optical microscope, and shown in FIG. 7. As canbe seen from the photograph, the length of the bacterium cultured in 1.0mM DTT medium wat shortened to 1/3 to 1/2 of the bacteria cultured in noDTT medium.

The DTT ribbon-shaped microfibrils produced in NA-added media wereobserved by the electron microscope and the atomic force microscope, andfound that the major axes (width) was small, e.g. 56 nm, 57 nm, 70 nm,etc., but the minor axes (thickness) were in the range of 10 to 100 nm.On the other hand, the ribbon-shaped microfibrils produced in no DTTadded medium had a major axis (width) of 82 nm, 107 nm, etc and a minoraxis (thickness) in the range of 10 to 100 nm, and significant variationwas not observed compared with DTT added medium concerning the minoraxis.

After culturing 40 days, the bacterial cellulose produced made into asheet, and properties of the sheets obtained mM DTT, 0.5 mM DTT or noDTT were measured similar to Example 1. The results are shown in Table 3

                  TABLE 3                                                         ______________________________________                                                     Mean           Mean          Mean                                   Thick- Thick- Young's Elastic Elongation Elongation                          DTT ness ness Modulus Modulus at Rupture at Rupture                           (mM) (μm) (μm) (GPa) (GPa) (%) (%)                                    ______________________________________                                        0.50 38      38      18.5   17.8   1.00   1.32                                   37  17.1  1.10                                                                40  18.8  1.87                                                                36  16.9  1.32                                                               1.0 54 45 15.4 16.2 0.97 1.70                                                  36  15.8  2.10                                                                37  18.5  2.07                                                                52  15.1  1.67                                                               0   25 38 11.8 12.4 1.82 1.80                                                  44  11.3  2.22                                                                51  14.1  1.53                                                                32  12.3  1.62                                                             ______________________________________                                    

As shown in Table 3, the sheet obtained by culturing in 0.5 mM DTT, 1.0mM DTT medium varied in its properties, and Young's modulus wasimportant compared with the sheet obtained by culuring in no DTT medium.

Example 4

Acetobacter pasteurianus FERM BP-4176 was cultured in agitation cultureat 180 rpm instead of static culture, and the culture solution andbacterial cellulose were withdrawn, and the shape of bacteria wasobserved by the optical microscope, the electron microscope and theatomic force microscope, similar to Example 1.

That is, four main culture media having the aforementioned compositionwere used, and nalidixic acid (NA) was added thereto in a concentrationof 0.10 mM, or 0.20 mM, except one medium to which NA was not added.

As a result, the length of the cellulose-producing bacteria increased.The ribbon-shaped microfibrils produced in NA-added media were observedby the electron microscope and the atomic force microscope, and foundthat the major axes (width) was great, e.g. 170 nm, 250 nm, etc., butvariation in the minor axes was not observed.

After culturing 14 days, the bacterial cellulose produced was made intoa sheet, and Young's modulus of the sheets were measured, similar toExample 1.

As a result, the sheets obtained by culturing in 0.1 mM NA medium and in0.2 mM NA medium varied in their properties, and Young's modulus wasimproved compared with the sheet obtained by culturing in no NA medium.

What is claimed is:
 1. An isolated and purified bacterial cellulosecomprising microfibrils having a thickness of 10 to 100 nm and a widthof 250 to 1000 nM.
 2. The bacterial cellulose of claim 1, which has awidth of 250 to 700 nm.
 3. The bacterial cellulose of claim 1, which hasa width of 250 to 600 nm.
 4. The bacterial cellulose of claim 1, whichhas a width of 170 to 1000 nm.
 5. The bacterial cellulose of claim 1,which has a width of 250 to 700 nm.
 6. The bacterial cellulose of claim1, which has a width of 250 to 600 nm.
 7. The bacterial cellulose ofclaim 1, wherein the microfibrils are ribbon-shaped.
 8. A method ofproducing the bacterial cellulose of claim 1, which comprises culturingcellulose-producing bacteria which produce the bacterial celluloseextracellularly in a culture medium containing a cell divisioninhibitor, and recovering the bacterial cellulose produced in theculture medium.
 9. The method of claim 8, wherein the cell divisioninhibitor is selected from the group consisting of chloramphenicol, aprotein synthesis inhibitor, an organic compound having β-lactamaseinhibiting ability, nalidixic acid, promidic acid, pipemidic acid,oxolinaic acid, ofloxacin and enoxacin.
 10. The method of claim 9,wherein the protein synthesis inhibitor is selected from the groupconsisting of tetracycline, puromycin and erythromycin.
 11. The methodof claim 9, wherein the organic compound having β-lactamase inhibitingability is thienamycin.
 12. The method of claim 8, wherein theconcentration of the cell division inhibitor in the culture medium is0.01 to 5 mM.
 13. The method of claim 8, wherein the bacteria areAcetobacter.
 14. The method of claim 8, wherein the bacteria areAcetobacter pasteurianus FERM BP-4176.
 15. The bacterial cellulose ofclaim 1, which has a width of 430 to 1000 nm.
 16. The bacterialcellulose of claim 1, which has a width of 590 to 1000 nm.
 17. Thebacterial cellulose of claim 1, which has a Young's modulus of 13 to 20GPa.
 18. The bacterial cellulose of claim 1, which has a Young's modulusof 16 to 20 Gpa.
 19. The bacterial cellulose of claim 1, which has awidth of 340 to 1000 nm.
 20. The bacterial cellulose of claim 1, whichhas a width of 340 to 700 nm.
 21. The bacterial cellulose of claim 1,which has a width of 340 to 600 nm.